NL8700089A - Medicaments for treating AIDS - contg. 2',3'-di:deoxy-thymidine or 2',3'-di:deoxy-thymidinene - Google Patents

Abstract

Medicaments for use in the treatment of AIDS and related diseases contain 2',3'-dideoxythymidine (I) or 2',3-dideoxythymidinene (II): The compsns. are formulated for parenteral, oral, rectal, vaginal, intranasal or topical admin. The unit dose is 0.1-100 mg/kg.

Description

4

Br / ar / 18-Rega

Therapeutic application of dideoxythymidine and dideoxythymidinein.

The invention relates to a new therapeutic agent for the treatment of AIDS and related diseases, and also to the preparation and application thereof.

AIDS, or acquired immunodeficiency syndrome, is a pandemic disease characterized by a high sensitivity to unusual malignancies and weakening of the immune system, accompanied by life-threatening infections. The causative agent of AIDS has been identified as a retrovirus called HTLV-III / LAV or "human T-cell lym-10 photropic virus type Ill / lymphademopathy-associated virus".

In the present disclosure, this retrovirus will be referred to as HIV or human immunodeficiency virus, as proposed in Science, 232, 1486 (1986). Recently, some other viruses related to HIV and causing AIDS-related diseases or states have also been described.

It is already known that 3'-azido-2 ', 3'-dideoxythymidine (azidothymidine, AZT, N3ddThd) is a potent inhibitor of HIV replication in lymphocyte cultures and that it will inhibit cells against the cytopathogenic effect of that virus 2ü protect at a concentration of 5-10 yuM (Mitsuya et al.,

Proc. Natl. Acad. Sci., U.S.A., 82, 7096-7100 (1985). Furthermore, it is known that 2r, 3'-dideoxyribosyl derivatives of purines (adenine, guanine, hypoxantine) and pyrimidines (thymine and cytosine) significantly suppress HIV-induced cytopathogenicity in vitro. The most potent derivative of this series is 21,3'-dideoxycytidine which will already provide full antiviral protection at a low concentration of 0.5-1.0 / µM (Mitsuya et al., Proc. Natl. Acad.

Sci., U.S.A., 82, 1911-1915 (1986). In addition, it has recently been found that an unsaturated derivative of 2 ', 3'-dideoxycytidine, namely 21,3'-didehydro-2', 3'-dideoxycytidine or 2 ', 3'-dideoxycytidinene, as well as 2', 3 ' -dideoxycytidine itself is a potent and selective inhibitor of HIV in vitro (Balzarini et al. Biochem. Biophys. Res. Commun., T4Q, 35 735-742, (1986)).

Ö70ÖO8 9 * - 2 -

In addition to this known matter, there is a need for other substances that act as potential HIV inhibitors. Therefore, the primary object of the invention is to provide such inhibitors, as well as therapeutic compositions containing these inhibitors.

In accordance with the invention, it has now been found that 21,3'-dideoxythymidine and an unsaturated derivative thereof, namely 2 ', 3'-dideoxythymidinene, are also potent and selective anti-HIV agents. Their selectivity and potency is comparable to that of 21,3'-dideoxycytidine and therefore they can be used advantageously in pharmaceutical compositions intended for treating AIDS patients. Potential use against diseases caused by HIV-related viruses is also within the scope of the invention.

21,3'-dideoxythymidine (ddThd) and 2 ', 3'-dideoxythymidinene (ddeThd) can be represented by the following chemical formulas I and II: 22 o <rNr (tNk

HO —I a HO—, A

xy ddThd ddeThd

I II

Λ i - 3 -

Both substances were already synthesized by Horwitz et al. In the 1960s (cf. J. Org. Chem., 32, 817-818 (1967) and Tetrahedron Letters, 1964, 2725-2727 and 1966, 1343-1346). Therefore, the compounds as such and their chemical synthesis are not part of the present invention.

In their action as anti-HIV agents, 2 *, 3'-dideoxythymidine and 2 ', 3'-dideoxythymidinene exhibit selectivity and potency comparable to that of 2', 3'-dideoxycytidine. Both substances, however, are less potent HlV inhibitors than azidothymidine. By comparison, 2 *, 3'-dideoxythymidinene appears to have a more potent action and 2 ', 3'-dideoxythymidine has a more selective action. In fact, the selective action of 21,3'-dideoxythymidine 15 is almost identical to that of azidothymidine.

The unsaturated derivative, 2 ', 3'-dideoxythymidinene is more toxic to uninfected host cells than the saturated derivative 21,3'-dideoxythymidine. Nevertheless, in parallel tests, the selectivity index of 2 ', 3'-20 dideoxythymidinene was found to be only slightly less than that of 2', 3'-dideoxycytidine.

It should be noted that the anti-HIV activity of 2 ', 3'-dideoxythymidine has been previously tested without demonstrating a potent effect (compare Mitsuya et al., Proc.

Natl. Acad. Sci., Ü.S.A. 83, 1911-1915 (1986)). According to this publication, the cytopathogenic activity of HIV in ATH8 cells was completely inhibited by 2 ', 3'-dideoxycytidine and 2', 3'-dideoxythymidine at a concentration of 0.00 µM and 20 µM, respectively. In contrast, experiments leading to the present invention showed that the same substances in MT-4 cells provide complete protection against HIV at a concentration of 0.2 µM and 1 µM, respectively. Contrary to the literature mentioned, 2 ', 3'-dideoxythymidine therefore appears to offer good prospects as an anti-HIV agent.

Therapeutic preparations containing 2 ', 3'-dideoxythymidine or 2', 3'-dideoxythymidinene as active ingredients. *

V

- 4 - part for treating AIDS in human practice, can be in the form of powders, suspensions, solutions, sprays, emulsions, ointments or creams and can be used for topical administration, for intranasal, rectal, vaginal and also for oral or parenteral (intravenous, intradermal, intramuscular, intrathecal, etc.) administration. Such preparations can be prepared by combining the active ingredient of formula (I) or (II) (eg by mixing, dissolving, etc.) with pharmaceutically acceptable neutral excipients (such as aqueous or non-aqueous solvents, stabilizers, emulsifiers, detergents, additives), and further with dyes and fragrances if desired. The concentration of the active ingredient in the therapeutic composition can vary widely between 0.1% and 100%, depending on the mode of administration. Furthermore, the dose of the active ingredient to be administered may vary between 0.1 mg and 100 mg per kg. body weight.

The anti-HIV properties of 2 ', 3'-dideoxy-thymidine and 2 1, 3'-dideoxythymidinene are documented by the following examples which should not be read in a limiting sense. Various other 21,3'-dideoxynucleosides and their 21,3'-unsaturated derivatives are used therein for comparison purposes.

In the examples, reference is made repeatedly to the accompanying drawings, in which: Figure 1 is a graphical representation of the test results on the inhibition of the cytopathogenic effect of HIV in MT-4 cells by various 2 ', 3'-dideoxynucleosides and their 2 3, Unsaturated Derivatives, Fig. 2 is a graphical representation of the test results on inhibition of virus antigen expression in HIV-infected MT-4 cells by the same series of 2 ', 3'-dideoxynucleosides and their 2' , 31-unsaturated derivatives.

In the examples and the drawings the following abbreviations are used:> "'· O fi * - ·" "v * ï? Φ £> - 5 - HIV - human immunodeficiency virus, as originally derived from a" pool " of AIDS patients; ddAdo - 2 ', 3'-dideoxyadenosine; ddThd - 2', 3'-dideoxythymidine; 5 ddCyd - 2 ', 3'-dideoxycytidine; ddürd - 2 *, 3'-dideoxyuridine; ddeAdo 21, 3'-dideoxyadenosinene; ddeThd-2 ', 3'-dideoxythymidinene; ddeCyd-21,3'-dideoxycytidinene; 10 ddeur-2 *, 31-dideoxyuridinene; MT-4 cells - cells of a human T4 lymphocyte cell line containing HTLV- I carries (compare Harada et al, Science, 229, 563-566 (1985)); HTLV-I - Type 1 of the human T-cell lymphotropic virus; 15 rpmi-140 - a culture medium for cell cultures developed in Roswell Park Memorial Institution and supplied by Gibco, Grand Island, NY, CJ.SA

It contains inorganic salts (NaCl, NaHCO3, -Na2HPO4r etc.), glucose, various amino acids and various vitamins; CCID50 ~ <3rd virus dose sufficient to infect 50% of the cell culture.

As for the chemical compounds, ddCyd and ddürd were obtained from Pharmacia, and ddAdo and ddThd were obtained from Calbiochem-Behring. The unsaturated derivatives were synthesized by reacting a 2'-deoxy-31,51-epoxy-nucleoside with potassium t-butoxide in dimethyl sulfoxide according to the method of Horwitz et al. (J. Org.

Chem., 32, 817-818 (1967)). The spectral data was consistent with those previously published.

The HIV was obtained from the National Cancer Institute, Bethesda, Md, USA, where it was harvested from the culture fluid of HIV-producing H9 cells, as described by Popovic et al. In Science 224, 497-500 (1984). .

This virus was stored at -70 ° C until used.

The virus titer, expressed in CCID50, was predetermined.

f "A"

v f .. v ··. · ς. cf - 6 -

The MT-4 cells were obtained from the Institut Pasteur in Paris, France.

Example 1

Inhibition of the cytopathogenic effect of HIV.

Four 21,3'-dideoxynucleosides and their 2 ', 3'-unsaturated derivatives were compared for their inhibitory effect on the cytopathogenic effect of HIV in MT-4 cells. The method is described by Mitsuya et al. In Proc. Natl. Acad. Sci. U.S.A., 82, 7076-7100 (1985).

The MT-4 cells were adjusted to a concentration of 5 x 105 cells / ml. using RPMI-1640 medium, supplemented with 10% fetal calf serum and antibiotics, then 2 ml aliquots. of the cell suspension were infected with 20tyul of the virus fluid (titer: 10 8 CCID 50/15 ml; final titer: 300 CCID 50 / well). For the control tests, MT-4 cells were quasi-infected with 20Qftl cell culture medium. The cells were incubated at 37 ° C. After 90 minutes of incubation, 1.8 ml of culture medium was added to each portion of the cell suspension. Various dilutions (100 µl each) of the compounds to be tested in cell culture medium were prepared in the cavities of a plastic microtiter plate 20. Then, 100ml of the HIV-infected or the quasi-infected cell suspension was added to each well and the cell cultures were incubated at 37eC. After 5 days, cell viability was assessed microscopically in a renatocytometer using the trypan blue exclusion method.

The results of the MT-4 cell assay are shown in Figure 1 of the drawing, in which the number of viable cells after 5 days is plotted against the concentration of the compounds to be tested. The black columns refer to tests with HIV-infected cells, while the white columns refer to tests with quasi-infected cells.

Figure 1 shows that ddeThd was most potent in protecting the cells from destruction by the f. ~ a η o β δ

** V W * · V

- 7 - "virus. At 0r04 μΜ, ddeThd fully protected the MT-4 cells, while the second most potent compound, ddCyd, required a concentration of 0.2 µM to give complete protection. The substance ddeThd provided approximately 45% protection at 0.008 yuM, at which concentration ddCyd had no inhibitory activity at all.

MetddThd achieved complete protection against the cytopathogenic activity of HIV at a concentration of 1 µM. The substance ddeCyd also protected the cells against HIV at a concentration of 1 / M. The other compounds were less potent in their anti-HIV activity, since ddA0o gave complete protection only at 25 µM, and ddürd provided 80% protection at 125 and 250, while cideAdo and ddeürd showed practically no protective effect.

Figure 1 (the white columns) further shows that the test compounds start to act cytotoxically at higher concentrations and that the compounds show great differences in this respect. For example, ddeThd inhibited the number of viable cells by 45% at a concentration of 1 µM, while ddCyd reduced the number of viable cells by 25% at a concentration of 5 µM.

The values of ED50 (the 50% antiviral active dose) and of TD50 (the 50% cytotoxic dose) for the 25 test compounds are shown in Table 1. It also contains the values of the selectivity index (SI), which i.e. the ratio of TD50 to ED50 * is shown.

Table 1 30

Link ED50 (uM) TD50 (uM) SI

ddAdo 2.5> 250> 100 ddeAdo> 125 9.5 <0.08 35 ddThd 0.20> 125> 625 ddeThd 0.010 1.2 120 ddCyd 0.046 9.1 198 ddeCyd 0.13 7.9 61 ddürd 48> 250> 5.2 ddeürd_> 125_27_ <0.22> 'ί089 \ - 8 -

Table 1 shows that ddThd exhibited the highest selectivity index. This compound was non-toxic at concentrations up to 125 µM. Subsequently followed were the substances ddeThd, ddCyd and ddeCyd, which were relatively toxic, as well as ddAdo and ddürd, where the ED50 was relatively low. The remaining substances ddeAdo and ddeürd had a negative value for the selectivity index.

Example 2 Inhibition of virus antigen expression.

The same compounds as in Example 1 were also compared for their inhibitory effect on the expression of virus antigens in HIV-infected MT-4 cells. This effect was determined by indirect immunofluorescence and laser flow cytofluorography using polyclonal antibodies from an AlDS patient as a probe. The results are shown in Figure 2 of the drawing, in which the percentage of fluorescent cells (ie the percentage of antigen-positive cells) are plotted against the concentration of the test compounds. The symbols used have the following meanings: Φ ddAdo O ddeAdo ddThd tir ddeThd I ddCyd Q ddeCyd 25 A ddürd ddeürd

The mean percentage of fluorescent cells in the absence of any test compound (positive control) was 60 + 5%, while the mean percentage of fluorescent cells in the quasi-infected cells (negative control) was 1.0%.

It can be seen from Figure 2 that the substance ddeThd caused complete inhibition of virus antigen expression at a concentration of 0.04 µM. This concentration was equal to that required for complete protection against the cytopathogenic activity of HIV. Similarly, ddCyd completely suppressed expression of the virus antigen at 0.2 µM, at which concentration it also provided complete protection against the cytopathogenic activity of the virus. As a rule, there was a clear correlation between the inhibitory activity of the compounds on BIV antigen production and the inhibitory activity on the cytopathogenic effect of HIV.

***** £ ‘8 9

Claims (3)

A therapeutic agent for use in the treatment of AIDS and related diseases, which agent contains 2 ', 3 1-dideoxythymidine or 2', 3'-dideoxythymidinene as active ingredient. A method for treating AIDS and related diseases, characterized in that 2 ', 3'-dideoxythymidine or 2', 3'-dideoxythymidine is administered to a patient suffering from AIDS or a related disease. 3. Use of 2 ', 3'-dideoxythymidine or 2', 3'-10 dideoxythymidinene for the preparation of a therapeutic agent effective against AIDS and related diseases. ****** «7ÖC0 & 9